The Sky Sports football pundit Andy Gray has been fired for "unacceptable and offensive behaviour" amid the furore over his comments about a female assistant referee and new footage of him making sexist comments and gestures towards a female co-presenter.

Gray, a key component of Sky's Premier League coverage for almost two decades, was initially suspended along with the anchor, Richard Keys, yesterday after a Sunday newspaper published a transcript of the pair discussing the female official Sian Massey during the match between Wolves and Liverpool.

The pair talked about Massey's appointment for the game on Saturday, speaking when they believed their microphones were switched off. They agreed that female officials "don't know the offside rule" and were "hopeless", with Keys adding: "I can guarantee you there'll be a big one today. Kenny [Dalglish] will go potty." Further unbroadcast footage then emerged of Gray discussing Massey's looks with a Sky Sports colleague, Andy Burton.

Gray was finally sacked after a video was posted on YouTube that showed him asking fellow presenter Charlotte Jackson to "tuck this in for me love" – a reference to his microphone pack – as he gestured down his trousers during a commercial break. As Gray and Keys burst out laughing, Jackson was shown trying to ignore the pair.

Sky Sports' managing director, Barney Francis, who had disciplined both Gray and Richard Keys for their comments, said it was this new evidence that made Gray's position untenable.

"Andy Gray's contract has been terminated for unacceptable behaviour. After issuing a warning, we have no hesitation in taking this action after becoming aware of new information today," he said.

Sky, concerned the episode would undermine carefully nurtured attempts to transform its public image, said the new evidence related to "an off-air incident that took place in December 2010" and "came to light after Andy Gray had already been subjected to disciplinary action for his comments of 22 January 2011". The former Everton and Aston Villa striker, believed to be on a seven-figure salary, is one of several public figures to have taken legal steps against another arm of Rupert Murdoch's media empire – the News of the World – in relation to phone-hacking.

Keys, whose private apology to Massey on Sunday was accepted by the 25-year-old assistant referee, is not expected to return to the screen until next week at the earliest. Burton was also dropped from his role as touchline reporter for the Carling Cup semi-final between Birmingham City and West Ham United.

In the first recording Keys commented about a newspaper column by the West Ham United vice-chair Karren Brady: "The game's gone mad," he said. "See charming Karren Brady complaining about sexism? Yeah. Do me a favour, love."

Brady said the comments made her "blood boil", while England captain Rio Ferdinand labelled them "prehistoric".

Massey, who unwittingly found herself at the centre of the storm and thrust on to the front page of several tabloids, today stood down from running the line at Crewe's Alexandra Stadium in an attempt to avoid a media circus.

Mike Riley, the general manager of Professional Game Match Officials (PGMO), the body responsible for professional referees, said: "Sian is an excellent professional who has unwittingly found herself in the middle of a story that has nothing to do with her competence as a match official. Sian only wants to be notable for her performance as an assistant referee and is keen for things to get back to normal so she can return to officiating as soon as possible.

"Sian has the full backing of PGMO and we hope she continues her development, which has shown excellent progress so far. It is important for PGMO to have talented people from all backgrounds getting involved in refereeing."